JP5762691B2 - Astaxanthin production method by fermentation - Google Patents

Description

  The present invention relates to a method for producing a carotenoid containing astaxanthin by microbial fermentation.

  Carotenoids are natural pigments useful as feed additives, food additives, pharmaceuticals, and the like. Examples of carotenoids include astaxanthin, canthaxanthin, zeaxanthin, β-cryptoxanthin, lycopene, β-carotene, phenicoxanthine, adonixanthin, echinone, astereudenone, and 3-hydroxyechinenone.

  Among carotenoids, astaxanthin is useful as a feed additive such as a body color improving agent for salmon, trout, red sea bream, etc., which are farmed fish, and an egg yolk improving agent for poultry. In addition, astaxanthin has high industrial value as a safe natural food additive and health food material.

  Adonixanthin and phenicoxanthin are expected to be used as feed additives, food additives, pharmaceuticals and the like, as in the case of astaxanthin, by establishing an industrial production method. Furthermore, β-carotene is used as a feed additive, food additive, pharmaceutical, etc., canthaxanthin is used as a feed additive, food additive, cosmetics, etc., zeaxanthin is used as a food additive, feed additive, etc. ing. In addition, lycopene, echinenone, β-cryptoxanthin, 3-hydroxyechinenone, asteroidenone and the like are expected to be used as feed additives, food materials, and the like. Known methods for producing these carotenoids include chemical synthesis methods, extraction methods from natural products, and production methods using microorganisms.

  On the other hand, as a chemical synthesis method of astaxanthin, for example, a method by conversion of β-carotene (Non-patent document 1) and a method of synthesizing from a C15 phosphonium salt (Non-patent document 2) are known. Astaxanthin produced by the chemical synthesis method is sold as a feed additive. Astaxanthin is present in fish such as red sea bream and salmon, and in crustaceans such as shrimp, crab and krill, and can be extracted from these.

  Examples of the production method of astaxanthin by microorganisms include, for example, a culture method using green algae Haematococcus pluvialis (Patent Document 1), a fermentation method using red yeast Phaffia rhodozyma (Patent Document 2), and Paracoccus (Paracoccus). A fermentation method using bacteria belonging to the genus (hereinafter sometimes referred to as “Paracoccus genus bacteria”) has been reported.

  Examples of bacteria belonging to the genus Paracoccus that produce astaxanthin include the E-396 strain and the A-581-1 strain (Patent Literature 3 and Non-Patent Literature 3). Other bacteria belonging to the genus Paracocccus that produce astaxanthin include Paracoccus marcusii MH1 strain (Patent Document 4), Paracoccus haeundaensis BC74171 strain (Non-Patent Document 4), Paracoccus bacterium N- 81106 strain (Patent Document 5) and Paracoccus sp. PC-1 strain (Patent Document 6).

  By the way, the above-mentioned method for producing carotenoid has several problems. For example, the chemical synthesis method gives an unfavorable impression to consumers from the viewpoint of safety. Extraction from natural products such as shrimp and crabs is expensive to manufacture. Furthermore, in production by green algae and yeast, carotenoids are difficult to extract due to low productivity and a strong cell wall.

  On the other hand, bacteria belonging to the genus Paracoccus have advantages such as high growth rate, high productivity, and easy extraction, and several culture methods have been reported. For example, Patent Document 7 discloses a method of adding an iron salt during culturing. Patent Document 8 discloses a method for limiting the carbon source concentration. However, these culture methods have a problem of accumulating a large amount of canthaxanthin simultaneously with astaxanthin.

  Canthaxanthin is useful as a feed additive to improve the color of salmon meat and chicken egg yolk, while in Europe, 0.03 mg / k-body weight is defined as ADI (permissible daily intake), The upper limit that can be added is 25 mg / kg for salmon and 8 mg / kg for laying hens (Non-patent Document 5). Therefore, when producing astaxanthin by microorganisms, it is necessary to keep the content of canthaxanthin low. Patent Document 9 discloses a method of reducing canthaxanthin production by controlling the dissolved oxygen concentration. However, this method is not practical from the viewpoint of manufacturing cost because the production concentration of astaxanthin is significantly reduced.

JP 2007-97584 A Japanese Patent Laid-Open No. 11-69969 Japanese Unexamined Patent Publication No. 7-79796 Special table 2001-512030 gazette JP 2007-244205 A International Publication No. 2005/118812 Pamphlet JP 2007-143492 A JP 2008-167665 A JP 2001-352995

Erich Widmer et al., "Pure Appl. Chem.", 1985, 57, pp.741-752 Erich Widmer et al., "Helv. Chim. Acta", 1981, Vol. 64, pp.2436-2446 Akira Tsubokura et al., "International Journal of Systematic Bacteriology", 1999, 49, pp.277-282 Jae Hyung Lee et al., `` International Journal of Systematic and Evolutionary Microbiology '', 2004, 54, pp.1699-1702 Official Journal of the European Communities L 22 / 28-30, 25.1.2003

  The present invention has been made in view of such a situation, and an object thereof is to provide a method for microbiologically producing astaxanthin at a high concentration and at a low cost while suppressing the production of canthaxanthin.

  As a result of various studies to solve the above-mentioned problems, the present inventors have found that in the cultivation of bacteria that simultaneously produce astaxanthin and canthaxanthin, biotin is added to the medium while maintaining the production concentration of canthaxanthin low. The inventors have found that it is possible to produce a concentration of astaxanthin, and have completed the present invention.

The present invention includes the following.
(1) including a step of culturing a bacterium that simultaneously produces astaxanthin and canthaxanthin in a biotin-containing medium, and compared with a culture after completion of culture in a biotin-free medium, canthaxanthin against astaxanthin in the culture after completion of culture A method for producing a carotenoid containing astaxanthin, characterized in that the production concentration ratio of the astaxanthin is low.
(2) The method according to (1), wherein the concentration of biotin per medium is 0.001 mg / L to 50 mg / L.
(3) The method according to (1), wherein the ratio of the production concentration of canthaxanthin to astaxanthin in the culture after completion of the culture is 25% by mass or less.
(4) The method according to (1), wherein the production concentration of gluconic acid in the culture after completion of the cultivation is 30 g / L or less.
(5) The method according to (1), wherein the dissolved oxygen concentration in the culture is controlled to 1 ppm or more in the culture.

(6) The method according to (1), wherein the content of poly-β-hydroxybutyric acid (hereinafter referred to as “PHB”) per dry cell in the culture after completion of the culture is 30% by mass or less.
(7) In the culture, the dissolved oxygen concentration in the culture is controlled to 1 ppm or more, and the ratio of the production concentration of canthaxanthin to astaxanthin in the culture after completion of the culture is 25% by mass or less, 1) The method described.
(8) In the culture, the dissolved oxygen concentration in the culture is controlled to 2 ppm or more, and the ratio of the production concentration of canthaxanthin to astaxanthin in the culture after completion of the culture is 8% by mass or less, 1) The method described.
(9) The method according to (1), wherein the concentration of dissolved oxygen in the culture is controlled stepwise or continuously in culture.
(10) Production of canthaxanthin relative to astaxanthin in the culture after completion of culture by controlling the initial dissolved oxygen concentration in the culture at the middle stage of culture to 1 to 2.5 ppm and increasing the dissolved oxygen concentration stepwise or continuously. The method according to (1), wherein the concentration ratio is 25% by mass or less.

(11) Production of canthaxanthin relative to astaxanthin in the culture after completion of the culture by controlling the initial dissolved oxygen concentration in the culture at the middle stage of culture to 2 to 3.5 ppm and increasing the dissolved oxygen concentration stepwise or continuously. The method according to (1), wherein the concentration ratio is 8% by mass or less.
(12) The method according to (1), wherein the bacterium belongs to the genus Paracoccus.
(13) The method according to (1), wherein the bacterium is a mutant strain having reduced PHB production ability.
(14) The method according to (1), wherein the bacterium is a mutant strain having a reduced ability to produce gluconic acid.
(15) The method according to (1), wherein the bacterium is a bacterium in which the base sequence of DNA corresponding to 16S ribosomal RNA is substantially homologous to the base sequence described in SEQ ID NO: 1.

(16) The method according to (15), wherein the bacterium is E-396 strain (FERM BP-4283), A-581-1 strain (FERM BP-4671) or a mutant thereof.
(17) A carotenoid composition for feed containing a carotenoid containing astaxanthin produced by the method according to (1), wherein the ratio of canthaxanthin to astaxanthin in the produced carotenoid is 25% by mass or less. And said composition.
(18) The carotenoid composition for feed according to (17), wherein the carotenoid containing astaxanthin has a PHB content of 30% by mass or less.
(19) A carotenoid composition for food containing a carotenoid containing astaxanthin produced by the method according to (1), wherein the ratio of canthaxanthin to astaxanthin in the produced carotenoid is 8% by mass or less. And said composition.

  According to the present invention, a high concentration of astaxanthin can be produced at low cost while keeping the concentration of canthaxanthin low in microbiological production. The carotenoids produced according to the present invention are useful as feed and food materials.

  Hereinafter, the present invention will be described in more detail. The scope of the present invention is not limited to these descriptions, and modifications other than the following examples can be made as appropriate without departing from the spirit of the present invention.

  The present invention is a method for producing a carotenoid containing astaxanthin by culturing a bacterium that simultaneously produces astaxanthin and canthaxanthin (hereinafter sometimes referred to as `` carotenoid-producing bacterium '' or `` astaxanthin-producing bacterium '') in a biotin-containing medium ( Hereinafter, it is referred to as “this method”. In this method, the ratio of the production concentration of canthaxanthin to astaxanthin in the culture after completion of the culture is lower than that in the culture of the same carotenoid-producing bacterium after completion of the culture in the biotin-free medium. In this method, by adding biotin to the medium, it is possible to produce astaxanthin at a high concentration at a low cost while suppressing the production concentration of canthaxanthin.

  The bacterium used in this method is not limited as long as it is a bacterium that simultaneously produces astaxanthin and canthaxanthin, but bacteria belonging to the genus Paracoccus are preferably used. Of the bacteria belonging to the genus Paracoccus, Paracoccus carotinifaciens, Paracoccus marcusii and Paracoccus haeundaensis are preferably used, and Paracoccus carotinifaciens is particularly preferably used. Specific examples of bacteria belonging to the genus Paracoccus include Paracoccus carotinifaciens E-396 (FERM BP-4283) and Paracoccus genus A-581-1 (FERM BP-4671) (Patent Document 3 and Non-Patent Documents) 3), and these strains are also preferably used in the present method.

  As the carotenoid-producing bacterium, preferably used is a bacterium whose DNA base sequence corresponding to 16S ribosomal RNA is substantially homologous to the base sequence of E-396 strain described in SEQ ID NO: 1. Here, “substantially homologous” means that the nucleotide sequence is preferably 95% or more, more preferably 96% or more, and still more preferably 97% in consideration of the error frequency when determining the DNA nucleotide sequence. Above, particularly preferably means 98% or more, most preferably 99% or more homology. The homology can be determined by, for example, gene analysis software Clustal W.

  The “base sequence of DNA corresponding to 16S ribosomal RNA” means a base sequence in which U (uracil) in the base sequence of 16S ribosomal RNA is replaced with T (thymine).

  In recent years, classification methods of microorganisms based on the homology of the base sequence of the 16S ribosomal RNA have become mainstream. The conventional classification method of microorganisms is based on the conventional bacteriological properties such as motility, auxotrophy, sugar utilization, etc. There was a case of misclassification. On the other hand, since the base sequence of 16S ribosomal RNA is extremely genetically stable, the classification method based on the homology greatly improves the classification reliability compared to the conventional classification method.

  The base sequence of 16S ribosomal RNA of Paracoccus carotinifaciens E-396 and other carotenoid-producing bacteria Paracoccus marcusii DSM 11574 (International Journal of Systematic Bacteriology (1998), 48, 543-548), Paracoccus genus N-81106 ( Patent document 5), Paracoccus haeundaensis BC 74171 strain (Non-patent document 4), Paracoccus genus A-581-1 strain and Paracoccus sp. PC-1 strain (patent document 6) between 16S ribosomal RNA base sequences The homology is 99.7%, 99.7%, 99.6%, 99.4%, and 95.4%, respectively, indicating that these strains are very closely related taxonomically. Therefore, it can be said that these strains form one group as bacteria producing carotenoids. For this reason, these strains are preferably used in this method, and can produce astaxanthin efficiently.

  In this method, a mutant strain with improved astaxanthin productivity can also be used. Examples of such mutants include mutants disclosed in JP-A-2001-95500, mutants disclosed in Patent Document 9, and the like.

  Alternatively, a mutant strain with improved astaxanthin productivity can be obtained by mutation treatment and screening. The mutation treatment method is not particularly limited as long as it induces mutation. For example, chemical methods using mutants such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonate (EMS), physical methods such as ultraviolet irradiation and X-ray irradiation, genetic recombination and Biological methods such as transposon can be used. In addition, the mutant strain may be generated by a naturally occurring mutation. From the viewpoint of public acceptance or safety, it is preferable to use a microorganism that is not a gene recombinant.

  The screening method for mutant strains with improved astaxanthin productivity is not particularly limited. For example, in addition to the method of selecting a target mutant strain by the color of colonies on an agar medium, test tubes, flasks, fermenters, etc. And a method of culturing the mutant strain in, and selecting the target mutant strain by carotenoid dye analysis using absorbance, high performance liquid chromatography, thin layer chromatography, or the like.

  The above-mentioned mutation treatment and screening steps may be performed once, or, for example, a mutant strain is obtained by mutation treatment and screening, and astaxanthin productivity is further improved by mutation treatment and screening of the obtained mutant strain. The mutation treatment and screening process may be repeated twice or more, such as obtaining a mutant strain.

  In this method, a mutant strain having a reduced ability to produce PHB (poly-β-hydroxybutyric acid) can be used. For example, the mutant strain can be derived from a bacterium belonging to the genus Paracoccus such as the aforementioned E-396 strain and A-581-1 strain. Bacteria that produce astaxanthin are known to accumulate PHB in cells as a storage carbon source. If PHB is accumulated, the medium carbon source will be consumed correspondingly, so it is better not to accumulate PHB as much as possible in order to reduce manufacturing costs. Therefore, it is effective to obtain a mutant strain with little or no PHB accumulation by mutation treatment and screening. As a specific method for obtaining a PHB low-producing strain, after carrying out the same mutation treatment as described above, for example, each mutant strain is cultured in a test tube, a flask, an agar medium, etc., and PHB is quantified. A method of selecting a mutant strain with a low production amount is mentioned.

  In the present method, a mutant strain having reduced gluconic acid production ability may be used. For example, the mutant strain can be derived from a bacterium belonging to the genus Paracoccus such as the aforementioned E-396 strain and A-581-1 strain. If gluconic acid is produced, the medium carbon source will be consumed correspondingly, and if a significant amount of gluconic acid is accumulated, growth and carotenoid production will be inhibited. Therefore, reducing the production of gluconic acid as much as possible is effective for the production of carotenoids. As a specific method for obtaining a gluconic acid low-producing strain, after carrying out the same mutation treatment as described above, for example, each mutant strain is cultured in a test tube, a flask, etc. A method of selecting a mutant strain having a small amount of gluconic acid by further measuring gluconic acid in a culture solution after selecting a mutant strain having a small decrease in the amount of gluconate.

  Mutants with favorable properties such as mutants with improved astaxanthin productivity, mutants with reduced PHB production ability, mutants with reduced gluconic acid production ability, etc. Although it is good, the mutation treatment and the screening can be repeated so that these two or more properties are combined. In addition, a mutant strain having two or more properties concomitantly obtained by combining two or more types of screening for one mutation treatment may be obtained. Mutants having these two or more preferred properties may be used in the present method.

  The E-396 strain mentioned as an example of a carotenoid-producing bacterium used in this method is deposited internationally as follows in the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

International Depositary Authority: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center
(Former name: Biotechnology Institute of Industrial Technology, Ministry of International Trade and Industry)
305-8566
Tsukuba City, Ibaraki Prefecture
Identification display: E-396
Accession Number: FERM BP-4283
Original deposit date: April 27, 1993

In addition, the A-581-1 strain mentioned as another example of the carotenoid-producing bacterium used in the present method has been deposited internationally with the above institution as follows.
Identification display: A-581-1
Accession number: FERM BP-4671
Original deposit date: May 20, 1994

  In addition to astaxanthin and canthaxanthin, the carotenoid produced by the present method is not particularly limited. For example, adonixanthin, phenicoxanthine, β-carotene, echinone, asteroidenone, 3-hydroxyechinenone, Zeaxanthin, β-cryptoxanthin, lycopene, preferably adonixanthin and adonilvin are mentioned. One kind of carotenoid produced by this method may be used, or a plurality of kinds may be combined.

  A method for culturing the bacteria in this method will be described below. In the following description, “culture” is not limited to a culture solution, but includes a solid, a semi-solid, and the like.

  The astaxanthin-producing medium used for cultivation in the present method may be any medium as long as it is a medium to which biotin is added and astaxanthin-producing bacteria grow and produce astaxanthin. A medium containing inorganic salts and, if necessary, vitamins is preferably used. That is, in this method, biotin is added to a medium in which astaxanthin-producing bacteria grow and can produce astaxanthin.

  Examples of the carbon source include sugars such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol and maltose, and organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid and pyruvic acid. , Alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol and glycenol, and fats and oils such as soybean oil, nuka oil, olive oil, corn oil, sesame oil and linseed oil, among which glucose or Sucrose is used. Among these carbon sources, one or more kinds can be used. The amount of the carbon source added to the medium before culture (starting medium) varies depending on the type of the carbon source and may be adjusted as appropriate, but is usually 1 to 100 g, preferably 2 to 50 g, per liter of the medium. In addition, the carbon source is preferably added not only to the starting medium, but also to be added sequentially or continuously during the culture.

  Examples of the inorganic nitrogen source include ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrates such as potassium nitrate, ammonia, urea, and the like. Among these, one or more kinds are used. . The addition amount varies depending on the type of nitrogen source and may be adjusted as appropriate, but is usually 0.1 to 20 g, preferably 0.2 to 10 g, with respect to 1 L of the medium.

  Examples of the organic nitrogen source include corn steep liquor (including filtered products), pharma media, soybean meal, soybean meal, peanut meal, sodium glutamate, distilers solver, and dry yeast. 1 type or 2 types or more are used. The addition concentration varies depending on the type of nitrogen source and may be adjusted as appropriate, but is usually 0 to 80 g / L, preferably 0 to 30 g / L in the medium.

  The inorganic nitrogen source and the organic nitrogen source are usually added to the starting medium, but it is also preferable to add them sequentially or continuously.

  Examples of inorganic salts include phosphates such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate, magnesium salts such as magnesium sulfate and magnesium chloride, iron salts such as iron sulfate and iron chloride, Calcium salts such as calcium chloride and calcium carbonate, sodium salts such as sodium carbonate and sodium chloride, manganese salts such as manganese sulfate, cobalt salts such as cobalt chloride, copper salts such as copper sulfate, zinc salts such as zinc sulfate, molybdic acid Molybdenum salts such as sodium, nickel salts such as nickel sulfate, selenium salts such as sodium selenate, boric acid and potassium iodide, and the like. Among these, one or more are used. The addition amount varies depending on the type of inorganic salt and may be adjusted as appropriate, but is usually 0.0001 to 15 g with respect to 1 L of the medium. For phosphates, magnesium salts, calcium salts, sodium salts and iron salts, the concentration in the medium is preferably 0.02 to 15 g / L. Manganese salts, cobalt salts, copper salts, zinc salts, molybdenum salts, nickel salts, selenium salts When boric acid, potassium iodide or the like is added, 0.1 to 15 mg / L is a preferable concentration. Inorganic salts are usually added to the starting medium, but may be additionally supplied sequentially or continuously.

  As vitamins other than biotin, for example, cyanocobalamin, riboflavin, pantothenic acid, pyridoxine, thiamine, ascorbic acid, folic acid, niacin, p-aminobenzoic acid, inositol, choline and the like can be used. The addition ratio varies depending on the type of vitamins and may be adjusted as appropriate, but is usually 0.001 to 1000 mg, preferably 0.01 to 100 mg, per 1 L of the medium. Vitamins are usually added to the starting medium, but may be added sequentially or continuously.

  The feature of this method is that astaxanthin-producing bacteria are cultured in a medium supplemented with biotin. By culturing astaxanthin-producing bacteria in a biotin-added medium, astaxanthin can be produced at a high concentration while keeping the canthaxanthin concentration low.

  The biotin used in this method may be DL-form or D-form, but preferably D-form is used. Biotin is usually added to the starting medium, but may be added intermittently or continuously during the culture, or may be added intermittently or continuously during the culture after addition to the starting medium. . Biotin may be sterilized by mixing with the main medium, or may be added after sterilizing separately. The biotin sterilization method is not particularly limited, and may be heat sterilization or filter sterilization.

  The concentration of biotin to be added per medium is not particularly limited, but is preferably 0.001 mg / L or more, more preferably 0.005 mg / L or more, further preferably 0.01 mg / L or more, particularly preferably 0.02 mg / L or more. It is. The biotin addition concentration is not particularly limited, but is preferably 50 mg / L or less, more preferably 20 mg / L or less, further preferably 10 mg / L or less, particularly preferably 5 mg / L or less, and most preferably 2 mg / L. L or less.

  In this method, an antifoaming agent is preferably used in order to suppress foaming of the culture. Any type of antifoaming agent may be used as long as it suppresses the generation of bubbles or has the effect of eliminating the generated bubbles and has little inhibitory effect on the produced bacteria. Examples thereof include alcohol-based antifoaming agents, polyether-based antifoaming agents, ester-based antifoaming agents, fatty acid-based antifoaming agents, silicon-based antifoaming agents, and sulfonic acid-based antifoaming agents. The addition amount of the antifoaming agent varies depending on the type of the antifoaming agent and may be adjusted as appropriate, but is usually 0.01 g to 10 g with respect to 1 L of the medium.

  Antifoam is usually added to the starting medium before sterilization. Furthermore, you may add an antifoamer continuously or intermittently during culture | cultivation. As a method of adding an antifoaming agent during the culture, for example, a method of automatically adding bubbles by sensing with a sensor, a method of adding by a program timer at regular intervals, a carbon source for feed, nitrogen to be linked to the growth rate Examples thereof include a method of adding a mixture with a source or a pH adjuster. The antifoaming agent added to the starting medium and the antifoaming agent added to the culture during the culture may be the same, but different types can be used according to the action.

  In this method, the pH of the medium at the start of the culture is adjusted to 2-12, preferably 6-9, more preferably 6.5-8.0. It is preferable to maintain the pH in the above range during the culture. As a method for maintaining the pH, a method in which the pH of the culture solution is measured online with a pH electrode installed in the fermenter and alkali is automatically supplied is preferable. Examples of the pH adjuster include an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, an aqueous sodium carbonate solution, ammonia water, ammonia gas, an aqueous sulfuric acid solution, or a mixture thereof.

  In this method, the medium is sterilized and then used for bacterial culture. A person skilled in the art can appropriately perform the sterilization treatment. For example, the medium in a suitable container may be heat sterilized with an autoclave. Or what is necessary is just to sterilize by filtration with a sterilization filter. Or what is necessary is just to sterilize by jacket heating and steam blowing. Carbon sources such as glucose turn brown when heat sterilized together with other medium components and may be sterilized separately. Vitamins and trace metals may be heat sterilized with the main medium, but may be sterilized separately to prevent inactivation and precipitation.

  In this method, astaxanthin-producing bacteria are inoculated into a biotin-added medium prepared as described above and cultured under predetermined conditions. Inoculation is performed by appropriately increasing the number of strains by seed culture using a test tube, flask, or fermenter, and adding the obtained culture to a biotin-added medium for producing astaxanthin. The medium used for seed culture is not particularly limited as long as it is a medium in which astaxanthin-producing bacteria grow well, whether it is a biotin-added medium or a biotin-free medium.

  Culture is performed in a suitable culture vessel. The culture vessel can be appropriately selected depending on the culture volume, and examples thereof include a test tube, a flask, and a fermenter.

  The culture temperature is, for example, 15 to 40 ° C, preferably 20 to 35 ° C, more preferably 25 ° C to 32 ° C. In addition, the culture period is usually 1 to 20 days, preferably 2 to 12 days, more preferably 3 to 9 days, particularly preferably 4 to 7 days, and culture is performed under aerobic conditions.

  Examples of aerobic conditions include shaking culture or aeration and agitation culture. However, if oxygen is deficient, the growth of astaxanthin-producing bacteria and carotenoid production are adversely affected, so the dissolved oxygen concentration is always monitored with a dissolved oxygen electrode. It is preferably done.

  By the way, since the number of microorganisms is small immediately after the start of culturing, the dissolved oxygen concentration shows a value close to the saturation concentration, but the dissolved oxygen concentration gradually decreases as the microorganism begins to grow and the oxygen consumption increases. The period until the dissolved oxygen concentration decreases to some extent, for example, 0 to 5 ppm, preferably 1 to 4 ppm, from the beginning of the culture to the initial stage of the culture, and then the period until the astaxanthin production concentration reaches the maximum value. The period from when the maximum value is reached until the end of the culture can be defined as the end of the culture.

  In the initial stage of the culture, the aeration amount, the number of rotations of stirring, and the pressure may be set low so that the dissolved oxygen concentration reaches the control region as soon as possible. However, in order to keep the mixed state of the culture well, a minimum number of rotations is necessary, and a minimum pressure is necessary to prevent contamination with germs.

  The middle culture period is the period when the oxygen consumption of microorganisms is most active. Here, if aeration and stirring are insufficient, the dissolved oxygen concentration reaches zero, that is, oxygen is depleted, which adversely affects the growth of microorganisms and the production of carotenoids. Therefore, it is preferable to control the dissolved oxygen concentration so that oxygen is not depleted in the middle of the culture. The dissolved oxygen concentration can be controlled, for example, by changing the rotation speed of stirring, the amount of ventilation, the internal pressure, the oxygen concentration in the aerated gas, and the like.

  In this method, in the culture of astaxanthin-producing bacteria, the higher the dissolved oxygen concentration in the culture, the lower the production concentration of canthaxanthin relative to astaxanthin, so the dissolved oxygen concentration in the culture in the middle of the culture is preferably It is controlled to 1 ppm or more, more preferably 1.5 ppm or more, further preferably 2 ppm or more, particularly preferably 2.5 ppm or more. The upper limit of the control range of the dissolved oxygen concentration in the culture in the middle of the culture is not particularly limited, but is preferably 8 ppm or less, more preferably 7 ppm or less, even more preferably 6 ppm or less, and particularly preferably 5 ppm or less.

  The dissolved oxygen concentration in the culture in the middle of the culture may be controlled to be constant, but increasing it stepwise or continuously is also effective for achieving a high astaxanthin concentration while suppressing the canthaxanthin concentration in the culture. . The number of steps is not particularly limited as long as it is 1 step or more, and may be increased semi-continuously or continuously, for example, by increasing 0.2 ppm per hour for 20 hours. The lower limit of the dissolved oxygen concentration (that is, the initial dissolved oxygen concentration) before increasing the dissolved oxygen concentration in the culture stepwise or continuously is not limited, but is preferably 1 ppm or more, more preferably 1.5 ppm or more, and still more preferably Is 2 ppm or more, and there is no upper limit, but it is preferably 3.5 ppm or less, more preferably 3 ppm or less, and even more preferably 2.5 ppm or less. In addition, the lower limit of the dissolved oxygen concentration after increasing the dissolved oxygen concentration in the culture stepwise or continuously is not limited, but is preferably 2.5 ppm or more, more preferably 3 ppm or more, and even more preferably 3.5 ppm or more. . The upper limit is not limited, but is preferably 8 ppm or less, more preferably 7 ppm or less, still more preferably 6 ppm or less, and particularly preferably 5 ppm or less.

  There is no particular limitation on the timing at which the dissolved oxygen concentration in the culture starts to increase stepwise or continuously in the middle of the culture, but it is preferably 0 to 60 hours, more preferably 2 to 50 hours from the beginning of the middle culture. The time is more preferably 4 hours to 40 hours, particularly preferably 6 hours to 30 hours. The time from when the dissolved oxygen concentration in the culture starts to increase stepwise or continuously until the highest dissolved oxygen concentration is reached is not limited. A one-step shift may reach the highest dissolved oxygen concentration within an hour. In the case of a semi-continuous or continuous increase of two or more stages, the time from the start of increasing the dissolved oxygen concentration in the culture to the maximum concentration is not limited, but preferably 2 hours to 120 hours. More preferably 4 hours to 100 hours, still more preferably 6 hours to 90 hours, particularly preferably 8 hours to 80 hours, and most preferably 10 hours to 70 hours.

  In addition, during the middle of the culture, the culture is sampled as needed, and while analyzing the carotenoid composition, the dissolved oxygen concentration in the culture is increased during the culture so that the ratio of the canthaxanthin concentration to the astaxanthin concentration becomes the desired value. The lowering can also be preferably performed. That is, when the canthaxanthin / astaxanthin ratio is higher than desired, the control setting value of the dissolved oxygen concentration in the culture is increased. On the other hand, when the canthaxanthin / astaxanthin ratio is low, the dissolved oxygen concentration in the culture is decreased. It is effective to do.

  At the end of the culture, the oxygen consumption activity of microorganisms decreases, and the production of bacterial cells and the production of carotenoids stop, so it is not necessary to control the dissolved oxygen concentration in the culture as strictly as in the middle of the culture, The dissolved oxygen concentration control similar to the latter period may be continued, or the culture may be performed with constant stirring and constant aeration.

  The ratio of the canthaxanthin concentration to the astaxanthin concentration in the culture finally obtained after completion of the culture after culturing by the above method is preferably 25% by mass or less, more preferably 20% by mass or less, and further preferably 15% by mass. % Or less, even more preferably 8% by weight or less, particularly preferably 6% by weight or less, and most preferably 4% by weight or less. Further, the ratio of the canthaxanthin concentration to the astaxanthin concentration in the culture after completion of the culture is not particularly limited, but is preferably 0.5% by mass or more, more preferably 1.0% by mass or more. A culture in which the ratio of canthaxanthin to astaxanthin in the culture after completion of the culture is 25% by mass or less can be suitably used for feed addition, and a culture having 8% by mass or less is suitable for foods. Can be used.

  Bacteria that produce astaxanthin and canthaxanthin simultaneously produce adonixanthin as a by-product. The ratio of the adonixanthin concentration in the culture obtained after culturing by the above method to the astaxanthin concentration is preferably 100% by mass or less, more preferably 90% by mass or less, and still more preferably 80% by mass or less. Even more preferably, it is 30% by mass or less, particularly preferably 25% by mass or less, and most preferably 20% by mass or less. Further, the ratio of the adonixanthin concentration to the astaxanthin concentration in the culture after the culture is not particularly limited, but is preferably 1% by mass or more, more preferably 5% by mass or more.

  The bacterium used in this method produces gluconic acid in the culture medium. If gluconic acid is produced, the medium carbon source will be consumed correspondingly, and if a significant amount of gluconic acid is accumulated, growth and carotenoid production will be inhibited. Therefore, reducing the production of gluconic acid as much as possible is effective for the production of carotenoids. In this method, the amount of gluconic acid produced can be reduced by adding biotin to the medium. The gluconic acid concentration in the finally obtained culture after completion of the culture is preferably 30 g / L or less, more preferably 20 g / L or less, still more preferably 10 g / L or less, and particularly preferably 5 g / L. L or less, and the lower limit is 0 g / L.

  In this method, the addition of biotin to the medium can suppress the production of PHB (poly-β-hydroxybutyric acid). The PHB content per dry cell in the finally obtained culture after completion of the culture is preferably 30% by mass or less, more preferably 20% by mass or less, still more preferably 10% by mass or less, particularly preferably 5%. The lower limit is 0% by mass. In particular, a culture having a PHB content per dry cell of 30% by mass or less in the culture after completion of the culture can be suitably used for feed addition.

  In this method, the carotenoid in the culture obtained by culturing astaxanthin-producing bacteria or the carotenoid collected from the culture can be quantified, for example, by high performance liquid chromatography.

  Astaxanthin-producing bacteria are cultured as described above, and the resulting culture may be used as it is as a carotenoid, or from a culture such as a culture solution, for example, a culture supernatant, a cell concentrate (cell concentrate) Alternatively, wet cells, dry cells, cell lysates, etc. may be prepared and these preparations may be used. Furthermore, carotenoids can be collected from these cultures or preparations by extraction, purification, or the like.

  The culture supernatant may be prepared by removing cells from the culture by centrifuging or filtering the culture. A bacterial cell concentrate (bacterial cell concentrate) can be obtained by centrifuging, centrifuging, or decanting the culture. Wet cells can be obtained by centrifuging or filtering the culture. The dried cells can be obtained by drying the culture, wet cells or cell concentrate (cell concentrate) by a general drying method. The carotenoid-containing dry cells thus obtained can be used as a feed additive as they are.

  In this method, the method for collecting carotenoid from the culture or preparation is not particularly limited, and any method in which carotenoid is stably and efficiently recovered may be used. Those skilled in the art can perform these methods by appropriately selecting from known extraction and purification techniques.

  Prior to extraction, the culture or preparation may be subjected to chemical treatment using an alkaline reagent or surfactant, biochemical treatment using a lytic enzyme, lipolytic enzyme, proteolytic enzyme, or the like, or ultrasound. Or you may use for one process or two or more processes in physical processes, such as a grinding | pulverization.

  For example, when carotenoids are extracted from a culture or preparation, the solvent used for extraction and washing is not particularly limited, but for example, lower alcohols such as methanol, ethanol, isopropanol, acetone, tetrahydrofuran, methyl ethyl ketone, methyl isobutyl ketone, Examples include dichloromethane, chloroform, dimethylformamide, dimethyl sulfoxide and the like.

  In order to prevent carotenoid oxidation during the extraction operation as much as possible, the treatment may be performed in an inert gas atmosphere such as nitrogen gas. Moreover, you may select the antioxidant used with a pharmaceutical and a foodstuff, and may add to an extraction solvent suitably. Alternatively, these processes may be combined.

  In order to prevent carotenoids from being decomposed by light as much as possible, extraction may be performed under conditions where no light is applied.

  The extract thus obtained can be used as it is as a carotenoid, and can also be used after further purification.

  Although it does not specifically limit as a method of isolate | separating bacteria etc. from extracts, such as an extract liquid after extraction operation, For example, membrane filtration, centrifugation, a decantation etc. are mentioned.

  As a method for obtaining a carotenoid precipitate from an extract, heating and / or vacuum concentration and crystallization are generally used. In addition, the carotenoid pigment may be separated without being concentrated by precipitation of the carotenoid pigment at a low temperature or by precipitation with an acid / alkali agent or various salts. In industrial use, it is desirable to crystallize.

  The obtained carotenoid precipitate may be suspended and stirred using a small amount of a solvent such as a lower alcohol as necessary for washing.

  Although the method of washing is not particularly limited, for example, a method of filtering after suspension and stirring, a method of passing liquid from above the precipitate, and the like are practically preferable methods.

  The cultures, preparations, extracts, or purified products obtained as described above can be used alone as carotenoids, or these can be mixed and used at an arbitrary ratio.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, the scope of the present invention is not limited to a following example.

  In addition, quantification of carotenoids in the examples was performed as follows using high performance liquid chromatography (HPLC).

  As the column, two Inertsil SIL-100A, 5 μm (φ4.6 × 250 mm) (manufactured by GL Science) were connected and used. Elution was performed by flowing 1.0 mL / min of a mobile phase n-hexane / tetrahydrofuran / methanol mixture (40: 20: 1) at a constant temperature near room temperature. In the measurement, the sample was dissolved in tetrahydrofuran and 20 μL of a 100-fold diluted solution in the mobile phase was used as the injection amount, and the column eluent was detected at a wavelength of 470 nm. As a standard for quantification, Sigma Astaxanthin (Cat. No. A9335) was used. The astaxanthin concentration of the standard solution was set using the following formula after measuring the absorbance at 477 nm of the standard solution (A) and the area percentage% (B) of the astaxanthin peak when HPLC analysis was performed under the above conditions. It was.

  Astaxanthin concentration (mg / L) = A ÷ 2150 × B × 100

[Example 1]
Medium of the following composition (sucrose 30 g / L, corn steep liquor 30 g / L, potassium dihydrogen phosphate 1.5 g / L, disodium hydrogen phosphate dodecahydrate 3.8 g / L, calcium chloride dihydrate 5.0 g / L, Magnesium sulfate heptahydrate 0.7 g / L, Iron sulfate heptahydrate 0.3 g / L, pH 7.2) 100 ml is placed in a 500 ml Erlenmeyer flask with a cotton stopper and autoclaved at 121 ° C for 15 minutes. Sterilized to prepare 7 seed flask media.

  Next, a medium with the following composition (glucose 20 g / L, corn steep liquor 30 g / L, ammonium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.25 g / L, disodium hydrogen phosphate dodecahydrate 5.7 g / L , Calcium chloride dihydrate 0.1g / L, magnesium sulfate heptahydrate 0.5g / L, iron sulfate heptahydrate 5g / L, alcohol antifoam 0.5g / L) 2.0L 5L fermentation Seven tanks were prepared in a tank. D-biotin was added to the fermenter to 0, 0.001, 0.01, 0.1, 1.0, 10 and 50 mg / L, respectively, and autoclaved at 121 ° C. for 30 minutes.

  Paracoccus carotinifaciens E-396 strain (FERM BP-4283) was inoculated into the above seed flask medium with one platinum ear, and cultured at 28 ° C for 2 days at 100 rpm. Inoculated into the fermentor. Aerobic culture at 28 ° C. and aeration volume of 1 vvm was performed for 120 hours. The pH was continuously controlled with 15% aqueous ammonia so that the pH was maintained at 7.2 during the culture. Glucose was added in an amount of 30 g on the first day, the second day, the third day, and the fourth day so as not to be depleted. In addition, the minimum stirring speed was set to 200 rpm, and the stirring speed was changed so that the dissolved oxygen concentration in the culture medium in the middle of the culture was maintained at 2 ppm. By detecting foaming with a bubble sensor, an alcohol-based antifoaming agent was automatically added to suppress foaming.

  When the carotenoid concentration, gluconic acid concentration, and PHB content per dry cell in the culture solution at the end of the culture were measured, the results were as shown in Table 1. It was found that the ratio of canthaxanthin to astaxanthin was lower in the experimental group to which biotin was added at 0.001 to 50 mg / L compared to the group to which biotin was not added.

[Example 2]
Medium of the following composition (sucrose 30 g / L, corn steep liquor 30 g / L, potassium dihydrogen phosphate 1.5 g / L, disodium hydrogen phosphate dodecahydrate 3.8 g / L, calcium chloride dihydrate 5.0 g / L, Magnesium sulfate heptahydrate 0.7 g / L, Iron sulfate heptahydrate 0.3 g / L, pH 7.2) 100 ml is placed in a 500 ml Erlenmeyer flask with a cotton stopper and autoclaved at 121 ° C for 20 minutes. Sterilized to prepare 8 seed flask media.

  Next, a medium having the following composition (sucrose 40 g / L, corn steep liquor 30 g / L, ammonium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.25 g / L, disodium hydrogen phosphate dodecahydrate 5.7 g / L L, calcium chloride dihydrate 0.1 g / L, magnesium sulfate heptahydrate 0.5 g / L, iron sulfate heptahydrate 5 g / L, alcohol-based antifoaming agent 0.5 g / L) Eight of these were prepared in a fermenter. D-biotin was added to the fermenter so that each concentration was 0.1 mg / L, and autoclaved at 121 ° C. for 30 minutes.

  Paracoccus carotinifaciens E-396 strain (FERM BP-4283) was inoculated into the above seed flask medium with one platinum ear, and cultured at 28 ° C for 2 days at 100 rpm. Inoculated into the fermentor. Aerobic culture at 28 ° C. and aeration volume of 1 vvm was performed for 120 hours. The pH was continuously controlled with 15% aqueous ammonia so that the pH was maintained at 7.2 during the culture. Sucrose was added in an amount of 30 g each on the first, second, third and fourth days of culture so as not to be depleted. The minimum stirring speed was 200 rpm, and the stirring speed was changed so that the dissolved oxygen concentration in the culture medium in the middle of the culture was maintained at 0.5, 1, 2, 3, 4, 5, 6, and 7 ppm. By detecting foaming with a bubble sensor, an alcohol-based antifoaming agent was automatically added to suppress foaming.

  When the carotenoid concentration, the gluconic acid concentration, and the PHB content per dry cell were measured at the end of the culture, the results are shown in Table 2. It was found that the higher the dissolved oxygen concentration, the lower the ratio of canthaxanthin concentration to astaxanthin concentration, which can be reduced to 1.4%.

  For comparison, the same experiment was performed in a medium not added with biotin. The results are shown in Table 3. The ratio of canthaxanthin concentration to astaxanthin concentration could only be reduced to 9%.

Example 3
Paracoccus carotinifaciens E-396 strain (FERM BP-4283) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to select colonies with deep red color. The PHB concentration and carotenoid concentration in the culture solution of the selected strain were measured, and a mutant LP-26 strain having a low PHB-producing ability and a high astaxanthin-producing ability was selected.

  Medium of the following composition (sucrose 30 g / L, corn steep liquor 30 g / L, potassium dihydrogen phosphate 1.5 g / L, disodium hydrogen phosphate dodecahydrate 3.8 g / L, calcium chloride dihydrate 5.0 g / L, Magnesium sulfate heptahydrate 0.7g / L, Iron sulfate heptahydrate 0.3g / L, pH7.2) 100ml is put into a 500ml Erlenmeyer flask with a cotton stopper and autoclaved at 121 ° C for 20 minutes Sterilized to prepare a seed flask medium.

  Next, a medium with the following composition (glucose 30 g / L, corn steep liquor 30 g / L, ammonium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.25 g / L, disodium hydrogen phosphate dodecahydrate 5.7 g / L , Calcium chloride dihydrate 0.1 g / L, magnesium sulfate heptahydrate 0.5 g / L, iron sulfate heptahydrate 5 g / L, sodium L-glutamate monohydrate 6 g / L, alcohol-based antifoaming agent 0.5L / 2.0) 2.0L was put into a 5L fermenter, and eight of them were prepared. D-biotin was added to the fermenter so that each concentration was 0.1 mg / L, and autoclaved at 121 ° C. for 30 minutes.

  The Paracoccus genus LP-26 strain selected above was inoculated into the seed flask medium with one platinum ear and subjected to rotary shaking culture at 28 ° C. for 2 days at 100 rpm. Inoculated in the fermenter. Aerobic culture was performed at 28 ° C. and aeration volume of 1 vvm for 140 hours. The pH was continuously controlled with 15% aqueous ammonia so that the pH was maintained at 7.2 during the culture. Glucose was added in an amount of 50 g on the first day, the second day, the third day, and the fourth day so as not to be depleted. Further, the minimum stirring speed was set to 100 rpm, and the stirring speed was changed so that the dissolved oxygen concentration in the culture medium in the middle of the culture was maintained at 0.5, 1, 2, 3, 4, 5, 6 and 7 ppm. By detecting foaming with a bubble sensor, an alcohol-based antifoaming agent was automatically added to suppress foaming.

  When the carotenoid concentration, the gluconic acid concentration, and the PHB content per dry cell were measured at the end of the culture, the results are shown in Table 4. It was found that the ratio of the canthaxanthin concentration to the astaxanthin concentration tended to decrease as the dissolved oxygen concentration was increased, and it could be reduced to 1.6%.

  For comparison, a similar experiment was performed by controlling the dissolved oxygen concentration in the middle of the culture to 2 ppm in a medium not added with biotin. The results are shown in Table 5. When biotin was added with the same dissolved oxygen concentration controlled at 2 ppm, the ratio of canthaxanthin to astaxanthin was 8%, whereas when biotin was not added, it was as high as 26.8%.

  Next, 0.1 mg / L of D-biotin was added, and the experiment other than the dissolved oxygen concentration was performed in the same manner as described above, and the dissolved oxygen concentration was shifted stepwise or continuously in the middle of the culture under the following four conditions. It was.

(Shift condition 1)
The initial dissolved oxygen concentration in the middle of the culture was controlled to 2 ppm and maintained at 2 ppm for 40 hours, then the control of the dissolved oxygen concentration was shifted to 2.5 ppm and maintained at 2.5 ppm until the end of the culture. That is, the dissolved oxygen concentration was reduced from the saturated concentration to 2 ppm after 0 to 8 hours from the start of the culture, controlled to 2 ppm for 8 to 48 hours, and 2.5 ppm for 48 to 140 hours.

(Shift condition 2)
Control the initial dissolved oxygen concentration in the middle of the culture to 1 ppm, maintain 1 ppm for 8 hours, increase the controlled concentration by 0.1 ppm per hour, increase the dissolved oxygen concentration to 5 ppm over 40 hours, and then until the end of the culture 5 ppm was maintained. That is, the dissolved oxygen concentration is reduced from the saturated concentration to 1 ppm after 0 to 9 hours from the start of culture, and is controlled to 1 ppm at 9 to 17 hours, and from 1 ppm to 5 ppm at 1 ppm from 17 to 57 hours per hour. Increased and controlled to 5 ppm for 57-140 hours.

(Shift condition 3)
After the initial dissolved oxygen concentration in the middle of the culture was controlled to 3 ppm and maintained at 3 ppm for 50 hours, the control of the dissolved oxygen concentration was shifted to 3.5 ppm and maintained at 3.5 ppm until the end of the culture. That is, the dissolved oxygen concentration was decreased from the saturated concentration to 3 ppm after 0 to 7 hours from the start of the culture, controlled to 3 ppm for 7 to 57 hours, and 3.5 ppm for 57 to 140 hours.

(Shift condition 4)
Control the initial dissolved oxygen concentration in the middle of the culture to 2 ppm, maintain 2 ppm for 4 hours, increase the controlled concentration by 0.1 ppm every 2 hours, increase the dissolved oxygen concentration to 7 ppm over 100 hours, and then until the end of the culture 7 ppm was maintained. That is, the dissolved oxygen concentration is reduced from the saturated concentration to 2 ppm at 0 to 8 hours after the start of the culture, controlled to 2 ppm at 8 to 12 hours, and from 12 ppm to 7 ppm at 0.1 ppm every 2 hours for 12 to 112 hours. Increased and controlled to 7 ppm for 112-140 hours.

  Culturing was performed under the above four conditions, and the carotenoid concentration, gluconic acid concentration, and PHB content per dry cell at the end of the culture (140 hours) were measured. The results are shown in Table 6. It was found that a culture solution having a high astaxanthin production concentration and a low canthaxanthin ratio can be obtained under any condition as compared with the case where the dissolved oxygen concentration was controlled to be constant (Table 4).

Example 4
Paracoccus carotinifaciens E-396 strain (FERM BP-4283) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to select colonies with deep red color. The selected strain was cultured in a test tube, and a mutant strain in which the pH of the culture broth was small and the culture broth was dark was selected. The gluconic acid concentration and carotenoid concentration in the test tube culture solution of the selected mutant strain were measured, and the mutant LG-7 strain having a low gluconic acid-producing ability and a high astaxanthin-producing ability was selected.

  Medium with the following composition (sucrose 30 g / L, Pharmamedia 10 g / L, potassium dihydrogen phosphate 0.8 g / L, dipotassium hydrogen phosphate 4.2 g / L, calcium chloride dihydrate 1 g / L, magnesium sulfate 100 ml of heptahydrate 12 g / L, iron sulfate heptahydrate 1 g / L, pH 7.2) is placed in a 500 ml Erlenmeyer flask with a cotton stopper, autoclaved at 121 ° C for 20 minutes, and the seed flask medium is Prepared.

  Next, a medium with the following composition (sucrose 30 g / L, Pharmamedia 20 g / L, ammonium sulfate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, disodium hydrogen phosphate 12 hydrate 3.8 g / L , Calcium chloride dihydrate 0.1g / L, Magnesium sulfate heptahydrate 4.5g / L, Iron sulfate heptahydrate 5g / L, Sodium L-glutamate monohydrate 6g / L, Silicone defoamer 1 g / L) 2.0 L was placed in a 5 L fermentor, and two of them were prepared. D-biotin was added to the fermenter to a concentration of 1 mg / L, and autoclaved at 121 ° C. for 30 minutes.

  The above-selected Paracoccus genus LG-7 strain was inoculated with one platinum ear in the seed flask medium, and cultured at 28 ° C for 3 days at 100 rpm. Inoculated in the fermenter. Aerobic culture at 28 ° C. and aeration volume of 1 vvm was performed for 120 hours. The pH was continuously controlled with 15% aqueous ammonia so that the pH was maintained at 7.2 during the culture. Sucrose was added in an amount of 40 g each on the 1st, 2nd, 3rd and 4th days of culture so as not to be depleted. In addition, the dissolved oxygen concentration in the culture broth was controlled under the following two conditions by setting the minimum stirring speed to 200 rpm and changing the stirring speed.

(Shift condition 5)
The initial dissolved oxygen concentration in the middle of the culture was controlled to 2.5 ppm and maintained at 2.5 ppm for 35 hours, and then the control of the dissolved oxygen concentration was shifted to 3 ppm and maintained at 3 ppm until the end of the culture. That is, the dissolved oxygen concentration was decreased from the saturated concentration to 2.5 ppm after 0 to 8 hours from the start of the culture, controlled to 2.5 ppm for 8 to 43 hours, and 3 ppm for 43 to 120 hours.

(Shift condition 6)
Control the initial dissolved oxygen concentration in the middle of the culture to 3.5 ppm, maintain 3.5 ppm for 4 hours, increase the controlled concentration by 0.1 ppm every 4 hours, increase the dissolved oxygen concentration to 5 ppm over 60 hours, and then culture Maintained 5 ppm until completion. That is, the dissolved oxygen concentration is decreased from the saturated concentration to 3.5 ppm at 0 to 7 hours after the start of the culture, and is controlled to 3.5 ppm at 7 to 11 hours, and from 3.5 ppm to 0.1 ppm at 11 to 71 hours for 4 hours. The amount was increased to 5 ppm each and controlled to 5 ppm for 71 to 120 hours.

  Culture was performed under the above two conditions, and the carotenoid concentration, gluconic acid concentration, and PHB content per dry cell at the end of the culture (120 hours) were measured. The results are shown in Table 7. For comparison, Table 7 shows the results of culture in which the dissolved oxygen concentration in the middle stage and the end stage of the culture was controlled under a constant condition of 4 ppm without adding biotin.

Example 5
Medium with the following composition (sucrose 20 g / L, corn steep liquor 5 g / L, potassium dihydrogen phosphate 0.54 g / L, dipotassium hydrogen phosphate 2.78 g / L, calcium chloride dihydrate 5 g / L, sulfuric acid 100 ml of magnesium heptahydrate 0.7 g / L, iron sulfate heptahydrate 3 g / L, pH 7.2) is placed in a 500 ml Erlenmeyer flask with a cotton stopper, autoclaved at 121 ° C for 20 minutes, and seed flask A medium was prepared.

  Next, a medium having the following composition (glucose 40 g / L, corn steep liquor 30 g / L, ammonium sulfate 0.5 g / L, potassium dihydrogen phosphate 2.25 g / L, disodium hydrogen phosphate dodecahydrate 5.7 g / L , Calcium chloride dihydrate 0.1 g / L, magnesium sulfate heptahydrate 0.5 g / L, iron sulfate heptahydrate 5 g / L, sodium L-glutamate monohydrate 6 g / L, alcohol-based antifoaming agent 0.5 g / L) 2.0 L was prepared in a 5 L fermenter. D-biotin was added to the fermenter to a concentration of 0.1 mg / L, and autoclaved at 121 ° C. for 30 minutes.

  Paracoccus genus A-581-1 strain (FERM BP-4671) was inoculated into the above seed flask medium by one platinum loop and cultured at 27 ° C for 2 days at 150 rpm. 80 mL was inoculated into the fermentor. Aerobic culture at 28 ° C. and aeration volume of 1 vvm was performed for 120 hours. The pH was continuously controlled with 15% aqueous ammonia so that the pH was maintained at 7.2 during the culture. Glucose was added in an amount of 30 g on the first day, the second day, the third day, and the fourth day so as not to be depleted. Further, the minimum stirring rotation speed was set to 200 rpm, and the dissolved oxygen concentration in the culture medium in the middle of the culture was controlled to 2 ppm by changing the stirring rotation speed.

  The carotenoid concentration at the end of the culture, the gluconic acid concentration, and the PHB content per dry cell were measured. The results are shown in Table 8. For comparison, Table 8 shows the results of culturing in which the dissolved oxygen concentration in the middle stage of culturing was controlled under a constant condition of 2 ppm without adding biotin.

Example 6
Paracoccus genus A-581-1 strain (FERM BP-4671) was mutated by ultraviolet irradiation to select colonies with deep red color. Carotenoids in the culture broth of the selected strain were analyzed, and a mutant K-185 strain with improved astaxanthin productivity was selected.

  Medium with the following composition (sucrose 30 g / L, corn steep liquor 30 g / L, potassium dihydrogen phosphate 1.5 g / L, disodium hydrogen phosphate 12 hydrate 3.8 g / L, calcium chloride dihydrate 5 g / L, Magnesium sulfate heptahydrate 0.7g / L, Iron sulfate heptahydrate 0.3g / L, pH7.2) 100ml in a 500ml Erlenmeyer flask with cotton stopper and autoclaved at 121 ° C for 20 minutes And a seed flask medium was prepared.

  Next, a medium having the following composition (glucose 30 g / L, soybean meal 20 g / L, ammonium sulfate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, disodium hydrogen phosphate dodecahydrate 3.8 g / L, Calcium chloride dihydrate 5g / L, Magnesium sulfate heptahydrate 0.7g / L, Iron sulfate heptahydrate 0.6g / L, Sodium L-glutamate monohydrate 6g / L, Ester defoamer 0.2 g / L) 2.0L was put in a 5L fermenter. D-biotin was added to the fermenter to a concentration of 1 mg / L, and autoclaved at 121 ° C. for 30 minutes.

  The above-selected Paracoccus genus K-185 strain was inoculated into a platinum flask in the above seed flask medium, and cultured at 27 ° C for 2 days at 150 rpm. Inoculated into the tank. Aerobic culture at 28 ° C. and aeration volume of 1 vvm was performed for 120 hours. The pH was continuously controlled with 15% aqueous ammonia so that the pH was maintained at 7.2 during the culture. Glucose was added in an amount of 30 g on the first day, the second day, the third day, and the fourth day so as not to be depleted. In addition, the dissolved oxygen concentration in the culture medium in the middle of the culture was controlled to 3.5 ppm by changing the minimum rotation speed to 200 rpm and changing the rotation speed.

  The carotenoid concentration at the end of the culture, the gluconic acid concentration, and the PHB content per dry cell were measured. The results are shown in Table 9. For comparison, Table 9 shows the results of culture in which the dissolved oxygen concentration in the middle stage of culture was controlled to 3.5 ppm without adding biotin.

Claims (8)

Bacteria belonging to the genus Paracoccus that produce astaxanthin and canthaxanthin at the same time are cultured in a medium containing biotin at a concentration of 0.005 to 50 mg / L but not yeast extract. In the culture, the dissolved oxygen concentration in the culture is controlled to 3 ppm or more in the culture, or the initial dissolved oxygen concentration in the culture is controlled to 2 to 3 ppm , and then the dissolved oxygen concentration in the culture is set to 3.5. A method for producing a carotenoid containing astaxanthin, wherein the ratio of the production concentration of canthaxanthin to astaxanthin in the culture after completion of the cultivation is stepwise or continuously increased to ppm or more and is 8.0% by mass or less. The method according to claim 1 , wherein the production concentration of gluconic acid in the culture after completion of the cultivation is 30 g / L or less. The method according to claim 1 , wherein the content of poly-β-hydroxybutyric acid (PHB) per dry cell in the culture after completion of the culture is 30% by mass or less. The method according to claim 1 , wherein the concentration of dissolved oxygen in the culture is controlled to increase stepwise or continuously at a timing of 2 to 50 hours from the beginning of the middle stage of culture. . The method according to claim 1 , wherein the bacterium is a mutant having a reduced PHB production ability. The method according to claim 1 , wherein the bacterium is a mutant having a reduced ability to produce gluconic acid. The method according to claim 1 , wherein the bacterium is a bacterium in which the base sequence of DNA corresponding to 16S ribosomal RNA is substantially homologous to the base sequence described in SEQ ID NO: 1. The method according to claim 1 , wherein the bacterium is E-396 strain (FERM BP-4283), A-581-1 strain (FERM BP-4671) or a mutant thereof.